PROJECT SUMMARY Drug craving during prolonged periods of abstinence is a major factor driving repeated cycles of drug abuse. In light of the increasing prevalence of drug abuse, it is imperative that we obtain a clear understanding of the neural circuit plasticity and associated molecular mechanisms underlying drug craving specifically. The ventral pallidum (VP) is the major output structure of the mesolimbic reward circuitry and is suggested to be the final common pathway for reward and motivational processing by relaying information from the nucleus accumbens (NAc) and ventral tegmental area (VTA) to the lateral habenula (LHb), VTA, and subthalamic structures. However, little is known about the circuit level organization and function of VP neurons in drug addiction, especially in the context of drug seeking following prolonged withdrawal. Therefore, we will anatomically and functionally probe the neural adaptations of a molecularly-defined subset of VP output neurons using the cocaine self-administration paradigm in mice, in order to better understand the mechanisms underlying cocaine seeking after a prolonged period of withdrawal. To accomplish this, we propose to study withdrawal-induced neural adaptations in specific subcircuits originating in the VP by using multiple cutting-edge techniques including optogenetic manipulation, in vivo monitoring of neural activity, viral-mediated tracing, ex vivo electrophysiology, and molecular profiling methods in a mouse cocaine self-administration model of drug addiction. Our preliminary data indicate that dopamine receptor 3 (Drd3) signaling is selectively upregulated in the VP during withdrawal from cocaine self-administration, and that knockdown of Drd3 in the VP, but not in the NAc, inhibits cocaine seeking behavior after prolonged withdrawal, but not sucrose reward seeking, strongly suggesting that VP Drd3 signaling may play a major role specifically in cocaine-induced craving and drug seeking behavior. We will first define the afferent and efferent connections of Drd3-expressing VP neurons. Second, we will examine the circuit- specific neural adaptations of VP Drd3 neurons and their role in cocaine seeking. Third, we will examine how VP Drd3 neuronal activity regulates VTA dopaminergic neuronal activity and dopamine release in NAc and VP during prolonged withdrawal from cocaine self-administration using cutting-edge imaging techniques. The accomplishment of this project will be greatly beneficial in providing a framework for studying drug addiction in a circuit-specific manner, as well as in developing a strategy for the treatment of drug addiction.